The hepatitis C virus (HCV) is the leading cause of liver disease in the United States. The currently employed interferon-based treatment is inadequate, as it has severe side effects and is only effective in half of the major genotype-infected individuals. The research proposed in this application will further the understanding of HCV cell entry mechanisms, with the ultimate goal of uncovering novel antiviral targets and developing additional systems to study this important human pathogen. With our recent discovery that two tight junction proteins, claudin-1 and occludin (OCLN), are essential for HCV entry, we have completed the list of cell type-specific HCV cell entry factors. Although we now know all the factors required for HCV cell entry and can essentially render any mammalian cell able to support this process, we do not understand how they mediate HCV infection. Here we propose studies to investigate the specific role for OCLN in this process. Initially we will examine OCLN specific HCV cell entry determinants though a comprehensive mutagenesis approach of this protein. To probe the functions of OCLN required for HCV cell entry, it is essential that we identify minimal mutations that dramatically influence its ability to act in this process. We will use nonfunctional OCLN variants as targets for the selection of mutant viruses with altered or expanded receptor usage abilities. Not only will this allow us to define genetic interactions between OCLN and HCV, but while doing so we may be able to generate a HCV mutants that can utilize murine OCLN, which would be a major advance towards the generation of a mouse adapted virus. We will use these virus and OCLN mutant sets to probe the mechanisms by which OCLN directs HCV cell entry. Essentially, we will survey a wide range of OCLN characteristics, from cellular localization, endocytosis, polarity-related, and protein-protein interaction abilities, to identify those that correlate with the capacity of each OCLN variant to function in the HCV entry process. These proposed experiments are directly related to the development of novel antiviral therapies to inhibit HCV cell entry. In addition, since OCLN appears to greatly influence the tight species tropism of HCV infection, this work may provide novel insights useful for the development of much-needed HCV small animal models.